Pituitary adenylate cyclase-activating polypeptide gene expression regulated by a testis-specific promoter in germ cells during spermatogenesis

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of t he glucagon-related family of hormones that is widely expressed in various tissues. The PACAP messenger RNA (mRNA) and protein is expressed at high le vels in the germ cells of the testis, where it locally activates cAMP-coupl ed receptors located in the somatic Sertoli cells. The PACAP mRNA expressed specifically in the testis is shorter than the mRNA expressed in hypothala mus and includes 127 nucleotides of novel sequence at the 5'-end, suggestin g a different start site of transcription in the testes and the utilization of a tissue-specific promoter. sere we present evidence that a single PACA P gene uses a testis-specific promoter to express a mRNA containing a uniqu e exon located 13.5 kb upstream from the first coding exon. As determined b y RT-PCR analysis of testis mRNA, the expression of the first testis-specif ic exon is relatively specific for the testis, as no PACAP mRNA containing the testis-specific first exon was detected in hypothalamic mRNAs. The prom oter for the testis-specific PACAP gene was cloned, and a start site for tr anscription was mapped by primer extension. The testis-specific promoter se quence directs germ cell-specific expression upon transfection of promoter- transcriptional reporter plasmids to populations of testicular cells in vit ro and upon expression of a promoter reporter transgene in mice. Analyses o f PACAP gene expression during the spermatogenic cycle, accomplished by RT- PCR of segments of isolated seminiferous tubules, identified intense expres sion in the postmeiotic round spermatids during developmental stages I-WI. These observations establish the existence of a specialized PACAP gene prom oter whose activity is highly regulated during the spermatogenic cycle.